Ropivacaine represses the ovarian cancer cell stemness and facilitates cell ferroptosis through inactivating the PI3K/AKT signaling pathway

Cell culture and treatment

Human ovarian cancer cell lines SKOV3 and OVCAR-3 were from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). All the cells were cultured in RPMI-1640 medium (Gibco, New York, USA) with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, USA) at 37°C, 5% CO₂.

SKOV3 and OVCAR-3 cells were treated with 0, 2.5, 5, 10, 20, 40, 80 and 160 μM doses of ropivacaine (BioChemPartner, Shanghai, China) for 24 h. ¹⁵ SKOV3 and OVCAR-3 cells were treated with 40 μM ropivacaine and/or 5 mmol/l erastin (MedChemExpress, New Jersey, USA), ¹⁶ 40 μM ropivacaine and/or 5 μM ferrostatin-1 (MedChemExpress) for 24 h. ¹⁷ SKOV3 cells were treated with 40 μM ropivacaine and/or 20 μM 740 Y-P (MedChemExpress) for 24 h. ¹⁸

Cell counting kit-8 (CCK-8) assay

SKOV3 and OVCAR-3 cells (2 × 10³) were inoculated in 96-well plates. After 24 h of culture, 10 μl CCK-8 solution (Beyotime, Shanghai, China) was added to each well and placed in an incubator for further culture for 1 h. Afterward, the 96-well plates were placed in a microplate reader (Thermo Fisher Scientific) and the absorbance (OD) at 450 nm was detected.

Flow cytometry

The ALDH positive cells were determined using an Aldefluor^TM kit (Stem Cell Technologies, Shanghai, China) referring to the reagent manufacturer’s procedure. SKOV3 and OVCAR-3 cells were incubated with AldefluorTM assay buffer containing ALDH substrate and BODIPY-Aminoacetaldehyde for 50 min at 37°C. Then the ALDH+ cells were delineated by catalyzing BODIPY-Aminoacetaldehyde to its fluorescent product BODIPY-Aminoacetate; the ALDH enzymatic activity was blocked by the specific ALDH inhibitor: AldefluorTM DEAB reagent. Ultimately, the ALDH positive cells were assessed using a flow cytometer (FACSCalibur, BD Biosciences, San Jose, USA) and the BD FACSAria II System (BD Biosciences).

Sphere-formation assay

SKOV3 and OVCAR-3 (1 × 10⁴) were inoculated in 96-well ultra-low attachment plates (Corning, NY, USA) and cultured in serum-free DMEM/F12 with the addition of 20 ng/mL human recombinant epidermal growth factor (Invitrogen, Carlsbad, USA), 10 ng/mL human recombinant basic fibroblast growth factor (Invitrogen) and antibiotics penicillin and streptomycin (Thermo Fisher Scientific) for 7 days. Then the sphere number was counted under a light microscope (Olympus, Tokyo, Japan) and Image J software (National Institutes of Health, Bethesda, USA).

Western blot

After SKOV3 and OVCAR-3 cells were harvested, the total proteins were extracted from the cells by RIPA buffer (Thermo Fisher Scientific). The protein concentrations were quantified using a BCA Assay Kit (Abcam, Cambridge, UK). The total amount of proteins (30 μg) were subjected to sodium dodecyl sulfonate-polyacrylamide gel electrophoresis (Thermo Fisher Scientific) and transferred onto polyvinylidene fluoride membranes (Millipore, Bedford, USA). After the membranes were blocked using 5% skim milk for 1 h, the membranes were incubated with the primary antibodies containing anti-OCT4 (Abcam, 1: 10,000, ab200834), anti-Nanog (Abcam, 1: 1000, ab109250), anti-GPX4 (Abcam, 1 µg/ml, ab41787), anti-SLC7A11 (xCT, Abcam, 1: 2000, ab175186), anti-p-PI3K (Abcam, 1: 500, ab182651), anti-PI3K (Abcam, 1: 1000, ab32089), anti-p-AKT (S473, Abcam, 1: 5000, ab81283), anti-p-AKT (T308, Abcam, 1: 500, ab38449), anti-AKT (Sigma-Aldrich, 1: 1000, SAB4500803), anti-p-mTOR (S2448, Abcam, 1: 1000, ab109268), anti-mTOR (Abcam, 1: 10,000, ab134,903) and anti-β-actin (Abcam, 1 µg/ml, ab8226) at 4°C overnight. The membranes were further incubated with the secondary antibody (Abcam) at room temperature for 1.5 h. The protein bands were assessed using enhanced chemiluminescence reagents (Millipore) and Image J software (National Institutes of Health).

Detection of the Fe²⁺ levels

The Fe²⁺ levels in ovarian cancer cells with different treatments were measured using an Iron Ion Kit (Millipore) following the reagent manufacturer’s procedure. In brief, the SKOV3 and OVCAR-3 cell supernatant was collected and incubated with an iron reducer, and then labeled with an iron probe. After that, an OD value of 590 nm was measured using a microplate reader (Thermo Fisher Scientific).

Immunofluorescence assay

The SKOV3 and OVCAR-3 cells with different treatments were harvested, washed with D-Hank’s buffer, and incubated with 2’,7’-dichlorofluorescein diacetate (DCFH-DA) at 37°C for 25  min. The DCF fluorescence of cells was analyzed using a fluorospectrophotometer at an excitation wavelength of 488 nm and an emission wavelength of 535 nm. The fluorescent intensity of DCF was tested by flow cytometry.

In vivo assay

Twelve Nude female BALB/c-nu mice (5-weeks-old) were from Shanghai Lab. Animal Research Center (Shanghai, China). All mice were kept in a temperature and humidity-controlled rooms with the 12 h light/dark cycle. All animal experiments were approved by the Animal Ethics Committee of Shandong Provincial Hospital Affiliated to Shandong First Medical University.

SKOV3 cells (5 × 10⁶) were injected subcutaneously into mice according to the previously described methods with minor changes. ¹⁹ To evaluate the effect of ropivacaine on the growth of ovarian cancer, ropivacaine (10 mg/kg) was injected intraperitoneally into mice referring to the previously reported methods with minor revisions. ¹⁵ The size of the tumor was measured every day and the tumor volumes were calculated by the formula: length × width²/2 = tumor volume (mm³). When the tumor size reached 2000 mm³, all mice were sacrificed and the excised tumor tissues were weighed to evaluate the antitumor effect.

Immunohistochemical assay

The xenografted tumor tissues were fixed with 10% formalin (Millipore) for 24  h, dehydrated and embedded in paraffin, and then made into 5 μm sections. The expressions of Ki-67, OCT4, Nanog, GPX4 and SLC7A11 were assessed using immunohistochemistry (IHC) methods with anti-Ki-67 (Abcam, 1 µg/ml, ab15580), anti-OCT4 (Abcam, 1: 250, ab200834), anti-Nanog (Abcam, 1: 100, ab109250), anti-GPX4 (Abcam, 1: 100, ab125066) and anti-SLC7A11 (Abcam, 1 µg/ml, ab37185) antibodies. Briefly, the sections were blocked by 1% BSA and were incubated with the above primary antibodies overnight at 4°C. Followed by the incubation with a secondary antibody for 1.5  h at room temperature. Subsequently, the sections were dyed with diaminobenzidine (DAB) and counterstained with hematoxylin. Ultimately, the sections were examined under a microscope (Olympus) and the percentage of positive cells was calculated.

Statistical analysis

All data were analyzed using SPSS 20.0 software (IBM, Chicago, USA). Data were presented as mean ± standard deviation. For the IC50 value of ropivacaine, the growth curve of ovarian cancer cells was drawn with different concentrations of ropivacaine as abscissa and cell viability (%) as ordinate. The IC50 value was the ropivacaine concentration of ovarian cancer cells at 50% viability. Differences between the two groups or more than two groups were assessed by Unpaired Student’s t-test or one-way ANOVA followed by Tukey’s post-test. p < .05 was considered statistically significant.

Ropivacaine restrains the ovarian cancer cell growth

The ovarian cancer cell growth was assessed after the SKOV3 and OVCAR-3 cells were treated with different doses of ropivacaine. The results demonstrated that the SKOV3 cell growth began to be repressed at a 10 μM dose of ropivacaine in comparison with the control group, and the IC50 was 18.64 μM (Figure 1(a)). Meanwhile, the growth of OVCAR-3 cells was also inhibited from the 10 μM dose of ropivacaine compared with the control group, and the IC50 was 24.86 μM ((Figure 1(b)). The 10, 20, and 40 μM doses of ropivacaine were selected for subsequent experiments referring to the experimental data of the CCK-8 assay. In general, ropivacaine repressed ovarian cancer cell growth.OPEN IN VIEWER

Ropivacaine represses the stemness of ovarian cancer cells

To further investigate the ropivacaine function on the ovarian cancer cell stemness, the ALDH positive cells were assessed after the ovarian cancer cells were treated with different concentrations of ropivacaine. As displayed in Figure 2(a), the ALDH positive cells were decreased gradually with the increased ropivacaine concentration, and the ALDH positive cells were reduced by nearly half at the 20 μM dose. As we all know, the sphere-formation ability is a pivotal method for the identification of tumor stem cells in vitro. Furthermore, the results of the sphere-formation assay confirmed that the sphere-formation ability of SKOV3 cells was weakened from the 10 μM dose of ropivacaine, and the sphere-formation ability of OVCAR-3 cells began to be reduced at a 20 μM dose of ropivacaine (Figure 2(b)). As presented in Figure 2(c), the protein levels of markers for stem cells OCT4 and Nanog protein levels began to be decreased in SKOV3 cells at a 10 μM dose of ropivacaine, and the OCT4 and Nanog protein levels in OVCAR-3 cells were down-regulated from the 20 μM dose of ropivacaine. The protein quantification results were displayed in Figure 2(c). To sum up, ropivacaine restrained the ovarian cancer cell stemness.OPEN IN VIEWER

Ropivacaine accelerates ferroptosis in ovarian cancer cells

Considering the importance of Fe²⁺ levels in ferroptosis (the novel mechanisms that regulate cell death and repress ovarian cancer cell growth), the Fe²⁺ levels were assessed in SKOV3 and OVCAR-3 cells and the results demonstrated that the Fe²⁺ levels were gradually elevated with the increased ropivacaine concentration and the Fe²⁺ levels increased nearly twofold when the ropivacaine dose was 20 μM (Figure 3(a)). The SKOV3 and OVCAR-3 cells were treated with ropivacaine and/or erastin (an agonist of ferroptosis), ropivacaine and/or ferrostatin-1 (inhibitor of ferroptosis). As displayed in Figure 3(b), the Fe²⁺ levels were further elevated after the erastin treatment, while the ferrostatin-1 treatment decreased the Fe²⁺ levels. Meanwhile, the ROS levels of SKOV3 cells were elevated from the 10 μM dose of ropivacaine, and the ROS levels of OVCAR-3 cells began to be increased at a 20 μM dose of ropivacaine (Figure 3(c)). Western blot data indicated that the protein levels of ferroptosis inhibitors GPX4 and SLC7A11 began to be decreased in SKOV3 cells at a 10 μM dose of ropivacaine, and the GPX4 and SLC7A11 protein levels in OVCAR-3 cells were reduced from the 20 μM dose of ropivacaine (Figure 3(d)). The above data suggested that ropivacaine accelerated the ferroptosis in ovarian cancer cells.OPEN IN VIEWER

Ropivacaine inactivates the PI3K/AKT signaling pathway in ovarian cancer cells

As reported, the abnormal activation of the PI3K/AKT signaling pathway accelerates ovarian cancer development. ²⁰ Therefore, we attempted to clarify whether ropivacaine participated in regulating the activation of the PI3K/AKT signaling pathway in ovarian cancer. Total-PI3K, total-AKT, and total-mTOR were internal controls for the calculation of relative expressions of p-PI3K, p-AKT (S473), p-AKT (T308) and p-mTOR (S2448), respectively. As displayed in Figure 4(a), the protein levels of p-PI3K and p-AKT (S473), p-AKT (T308), and p-mTOR (S2448) in cells were gradually decreased with the increase of ropivacaine concentration, while the total-PI3K, total-AKT and total-mTOR protein levels did not change significantly. After the SKOV3 cells were treated with ropivacaine and/or 740 Y-P (a commonly used PI3K agonist), the protein levels of p-PI3K and p-AKT (S473), p-AKT (T308), and p-mTOR (S2448) in cells were down-regulated after the ropivacaine treatment, while the 740 Y-P treatment partially reversed this down-regulation; the total-PI3K, total-AKT, and total-mTOR protein levels were unchanged between all groups (Figure 4(b)). Meanwhile, the CCK-8 results corroborated that the ropivacaine treatment reduced cell viability, and this reduction was partially reversed by the 740 Y-P treatment (Figure 4(c)). As displayed in Figure 4(d), the sphere-formation ability of ovarian cancer cells was weakened after the ropivacaine treatment, and this trend was partially reversed by the 740 Y-P treatment. Furthermore, the treatment of ropivacaine increased the Fe²⁺ levels, and the Fe²⁺ levels were increased nearly threefold, while the treatment of 740 Y-P partially reversed this increase (Figure 4(e)). Similarly, the ROS levels of ovarian cancer cells were elevated after the ropivacaine treatment, and this effect was partially reversed by the 740 Y-P treatment (Figure 4(f)). In summary, ropivacaine inactivated the PI3K/AKT signaling pathway in ovarian cancer cells.OPEN IN VIEWER

Ropivacaine restrains the proliferation of ovarian cancer cells in vivo

Subsequently, we further evaluated the function of ropivacaine in ovarian cancer in vivo. The ropivacaine treatment reduced the tumor volume in comparison with the sham group (Figure 5(a)) and the representative macroscopic figures of tumors were provided in Supplementary Figure 1. Also, the treatment of ropivacaine had no significant changes in the weight of mice in comparison with the sham group (Figure 5(b)). Meanwhile, the ropivacaine treatment decreased the tumor weight compared with the sham group (Figure 5(c)). Besides, the expressions of Ki-67 (proliferating marker), OCT4, Nanog, GPX4, and SLC7A11 in subcutaneous tumor tissues of mice were measured and the results confirmed that the ropivacaine treatment reduced the Ki-67, OCT4, Nanog, GPX4 and SLC7A11 expressions in tissues (Figure 5(d)). The above data suggested that ropivacaine weakened the proliferation of ovarian cancer cells in vivo.OPEN IN VIEWER